Comparing the Potential of Silicon Nanoparticles and Conventional Silicon for Salinity Stress Alleviation in Soybean (Glycine max L.): Growth and Physiological Traits and Rhizosphere/Endophytic Bacterial Communities.

In this study, 20-day-old soybean plants were watered with 100 mL of 100 mM NaCl solution and sprayed with silica nanoparticles (SiO2 NPs) or potassium silicate every 3 days over 15 days, with a final dosage of 12 mg of SiO2 per plant. We assessed the alterations in the plant's growth and physiological traits, and the responses of bacterial microbiome within the leaf endosphere, rhizosphere, and root endosphere. The result showed that the type of silicon did not significantly impact most of the plant parameters. However, the bacterial communities within the leaf and root endospheres had a stronger response to SiO2 NPs treatment, showing enrichment of 24 and 13 microbial taxa, respectively, compared with the silicate treatment, which led to the enrichment of 9 and 8 taxonomic taxa, respectively. The rhizosphere bacterial communities were less sensitive to SiO2 NPs, enriching only 2 microbial clades, compared to the 8 clades enriched by silicate treatment. Furthermore, SiO2 NPs treatment enriched beneficial genera, such as Pseudomonas, Bacillus, and Variovorax in the leaf and root endosphere, likely enhancing plant growth and salinity stress resistance. These findings highlight the potential of SiO2 NPs for foliar application in sustainable farming by enhancing plant-microbe interactions to improve salinity tolerance.

Structure modification of an antibiotic: by engineering the fusaricidin bio-synthetase A in Paenibacillus polymyxa.

Fusaricidin, a lipopeptide antibiotic, is specifically produced by Paenibacillus polymyxa strains, which could strongly inhibit Fusarium species fungi. Fusaricidin bio-synthetase A (FusA) is composed of six modules and is essential for synthesizing the peptide moiety of fusaricidin. In this study, we confirmed the FusA of Paenibacillus polymyxa strain WLY78 involved in producing Fusaricidin LI-F07a. We constructed six engineered strains by deletion of each module within FusA from the genome of strain WLY78. One of the engineered strains is able to produce a novel compound that exhibits better antifungal activity than that of fusaricidin LI-F07a. This new compound, known as fusaricidin [ΔAla6] LI-F07a, has a molecular weight of 858. Our findings reveal that it exhibits a remarkable 1-fold increase in antifungal activity compared to previous fusaricidin, and the fermentation yield reaches ~55 mg/L. This research holds promising implications for plant protection against infections caused by Fusarium and Botrytis pathogen infection.

Glutamine synthetase and GlnR regulate nitrogen metabolism in Paenibacillus polymyxa WLY78.

Paenibacillus polymyxa WLY78, a N2-fixing bacterium, has great potential use as a biofertilizer in agriculture. Recently, we have revealed that GlnR positively and negatively regulates the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) in P. polymyxa WLY78 by binding to two loci of the nif promoter according to nitrogen availability. However, the regulatory mechanisms of nitrogen metabolism mediated by GlnR in the Paenibacillus genus remain unclear. In this study, we have revealed that glutamine synthetase (GS) and GlnR in P. polymyxa WLY78 play a key role in the regulation of nitrogen metabolism. P. polymyxa GS (encoded by glnA within glnRA) and GS1 (encoded by glnA1) belong to distinct groups: GSI-α and GSI-ß. Both GS and GS1 have the enzyme activity to convert NH4+ and glutamate into glutamine, but only GS is involved in the repression by GlnR. GlnR represses transcription of glnRA under excess nitrogen, while it activates the expression of glnA1 under nitrogen limitation. GlnR simultaneously activates and represses the expression of amtBglnK and gcvH in response to nitrogen availability. Also, GlnR regulates the expression of nasA, nasD1D2, nasT, glnQHMP, and glnS. IMPORTANCE In this study, we have revealed that Paenibacillus polymyxa GlnR uses multiple mechanisms to regulate nitrogen metabolism. GlnR activates or represses or simultaneously activates and inhibits the transcription of nitrogen metabolism genes in response to nitrogen availability. The multiple regulation mechanisms employed by P. polymyxa GlnR are very different from Bacillus subtilis GlnR which represses nitrogen metabolism under excess nitrogen. Both GS encoded by glnA within the glnRA operon and GS1 encoded by glnA1 in P. polymyxa WLY78 are involved in ammonium assimilation, but only GS is required for regulating GlnR activity. The work not only provides significant insight into understanding the interplay of GlnR and GS in nitrogen metabolism but also provides guidance for improving nitrogen fixation efficiency by modulating nitrogen metabolism.

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy.

Nitrogenase in some bacteria and archaea catalyzes conversion of N2 to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of nif (nitrogen fixation) genes. Viral 2A peptide mediated "cleavage" of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in Saccharomyces cerevisiae ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

Genome-wide mapping of GlnR-binding sites reveals the global regulatory role of GlnR in controlling the metabolism of nitrogen and carbon in Paenibacillus polymyxa WLY78.

BACKGROUND: Paenibacillus polymyxa WLY78 is a Gram-positive, endospore-forming and N2-fixing bacterium. Our previous study has demonstrated that GlnR acts as both an activator and a repressor to regulate the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) according to nitrogen availability, which is achieved by binding to the two GlnR-binding sites located in the nif promoter region. However, further study on the GlnR-mediated global regulation in this bacterium is still needed. RESULTS: In this study, global identification of the genes directly under GlnR control is determined by using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assays (EMSA). Our results reveal that GlnR directly regulates the transcription of 17 genes/operons, including a nif operon, 14 nitrogen metabolism genes/operons (glnRA, amtBglnK, glnA1, glnK1, glnQHMP, nasA, nasD1, nasD2EF, gcvH, ansZ, pucR, oppABC, appABCDF and dppABC) and 2 carbon metabolism genes (ldh3 and maeA1). Except for the glnRA and nif operon, the other 15 genes/operons are newly identified targets of GlnR. Furthermore, genome-wide transcription analyses reveal that GlnR not only directly regulates the expression of these 17 genes/operons, but also indirectly controls the expression of some other genes/operons involved in nitrogen fixation and the metabolisms of nitrogen and carbon. CONCLUSION: This study provides a GlnR-mediated regulation network of nitrogen fixation and the metabolisms of nitrogen and carbon.

Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27.

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Biofertilization containing Paenibacillus triticisoli BJ-18 alters the composition and interaction of the protistan community in the wheat rhizosphere under field conditions.

AIMS: Most studies focus on the effects of biofertilizer on the bacterial and fungal communities, and we still lack an understanding of biofertilizer on the protistan community. Here, the effects of biofertilizer containing Paenibacillus triticisoli BJ-18 on composition and interaction of the protistan community in the wheat rhizosphere were investigated. METHODS AND RESULTS: Biofertilizer application altered soil physicochemical properties and the protistan community composition, and significantly induced an alpha diversity decline. Random forecast and redundancy analysis demonstrated that nitrogenase activity and available phosphorus were the main drivers. Trichomonas classified to the phylum Metamonada was enriched by biofertilizer, and was significantly positive connected with soil nitrogenase activity and some function genes involved in nitrogen-fixation and nitrogen-dissimilation. Biofertilization loosely connected biotic interactions, while it did not affect the stability of the protistan community. Besides, biofertilizer promoted the connections of protists with fungi, bacteria, and archaea. Combined with biotic networks (protists, fungi, bacteria, and archaea) and interactions between protists and soil physicochemical properties/function genes, protists may act as keystone taxa potentially driving soil microbiome composition and function. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, these results suggest that the biofertilizer is a driver of the soil protistan community, contributing to ecosystem functioning.

Paenibacillus caui sp. nov., a nitrogen-fixing species isolated from the rhizosphere soil of a peach tree.

A nitrogen-fixing, endospore-forming, motile, rod-shaped, facultative aerobic bacterium, designated 81-11T, was isolated from rhizosphere soil of a peach tree collected from Handan, Hebei, PR China. From the comparison of 16S rRNA gene sequence, the strain is most closely related to Paenibacillus phoenicis DSM 27463T (96.9 %) and Paenibacillus faecis DSM 23593T (96.7 %). The genome size of strain 81-11T was 4.4 Mb, comprising 4879 predicted genes with a DNA G+C content of 50.0 mol%. The average nucleotide identity values of genome sequences between the novel isolate and the type strains of related species P. phoenicis DSM 27463T and P. faecis DSM 23593T were 71.8 and 72.1 %, respectively. The major cellular fatty acids were anteiso-C15 : 0(47.8 %), iso-C16 : 0 (15.5 %) and iso-C15 : 0 (13.0 %). Menaquinone-7 was the major respiratory quinone. The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, aminophospholipid, aminoglycopid, unknown polar lipids and unidentified aminophosphoglycolipid. Based on phylogenetic, genomic and phenotypic characteristics, strain 81-11T was classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus caui sp. nov. is proposed. The type strain of Paenibacillus caui is 81-11T (=JCM 34618T=CGMCC 1.18907T).

Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres.

Two strains HN-1T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 99.9 and 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1T with P. stellifer DSM 14472T was 90.69, which was below the recommended threshold value (< 95-96% ANI). The dDDH showed 42.1% relatedness between strain HN-1T and P. stellifer DSM 14472T, which was lower than the recommended threshold value (dDDH < 70%). The strain HN-1T contain anteiso-C15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472T, strain HN-1T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.

Fusaricidin Biosynthesis Is Controlled via a KinB-Spo0A-AbrB Signal Pathway in Paenibacillus polymyxa WLY78.

Fusaricidins produced by Paenibacillus polymyxa are important lipopeptide antibiotics against fungi. The fusGFEDCBA (fusaricidin biosynthesis) operon is responsible for synthesis of fusaricidins. However, the regulation mechanisms of fusaricidin biosynthesis remain to be fully clarified. In this study, we revealed that fusaricidin production is controlled by a complex regulatory network including KinB-Spo0A-AbrB. Evidence suggested that the regulator AbrB represses the transcription of the fus gene cluster by direct binding to the fus promoter, in which the sequences (5'-AATTTTAAAATAAATTTTGTGATTT-3') located from -136 to -112 bp relative to the transcription start site is required for this repression. Spo0A binds to the abrB promoter that contains the Spo0A-binding sequences (5'-TGTCGAA-3', 0A box) and in turn prevents the further transcription of abrB. The decreasing concentration of AbrB allows for the derepression of the fus promoter repressed by AbrB. The genome of P. polymyxa WLY78 contains two orthologs (named Kin1508 and Kin4833) of Bacillus subtilis KinB, but only Kin4833 activates sporulation and fusaricidin production, indicating that this kinase may be involved in phosphorylating Spo0A to initiate sporulation and regulate the abrB transcription. Our results reveal that Kin4833 (KinB), Spo0A, and AbrB are involved in regulation of fusaricidin production and a signaling mechanism that links fusaricidin production and sporulation.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional analysis of multiple nifB genes of Paenibacillus strains in synthesis of Mo-, Fe- and V-nitrogenases.

BACKGROUND: Biological nitrogen fixation is catalyzed by Mo-, V- and Fe-nitrogenases that are encoded by nif, vnf and anf genes, respectively. NifB is the key protein in synthesis of the cofactors of all nitrogenases. Most diazotrophic Paenibacillus strains have only one nifB gene located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV). But some Paenibacillus strains have multiple nifB genes and their functions are not known. RESULTS: A total of 138 nifB genes are found in the 116 diazotrophic Paenibacillus strains. Phylogeny analysis shows that these nifB genes fall into 4 classes: nifBI class including the genes (named as nifB1 genes) that are the first gene within the compact nif gene cluster, nifBII class including the genes (named as nifB2 genes) that are adjacent to anf or vnf genes, nifBIII class whose members are designated as nifB3 genes and nifBIV class whose members are named as nifB4 genes are scattered on genomes. Functional analysis by complementation of the ∆nifB mutant of P. polymyxa which has only one nifB gene has shown that both nifB1 and nifB2 are active in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Deletion analysis also has revealed that nifB1 of Paenibacillus sabinae T27 is involved in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Complementation of the P. polymyxa ∆nifBHDK mutant with the four reconstituted operons: nifB1anfHDGK, nifB2anfHDGK, nifB1vnfHDGK and nifB2vnfHDGK, has shown both that nifB1 and nifB2 were able to support synthesis of Fe- or V-nitrogenases. Transcriptional results obtained in the original Paenibacillus strains are consistent with the complementation results. CONCLUSIONS: The multiple nifB genes of the diazotrophic Paenibacillus strains are divided into 4 classes. The nifB1 located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV) and the nifB2 genes being adjacent to nif or anf or vnf genes are active in synthesis of Mo-, Fe and V-nitrogenases, but nifB3 and nifB4 are not. The reconstituted anf system comprising 8 genes (nifBanfHDGK and nifXhesAnifV) and vnf system comprising 10 genes (nifBvnfHDGKEN and nifXhesAnifV) support synthesis of Fe-nitrogenase and V-nitrogenase in Paenibacillus background, respectively.

Identification of Genes Involved in Fe-S Cluster Biosynthesis of Nitrogenase in Paenibacillus polymyxa WLY78.

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe-S cluster in diazotrophs. However, nifS and nifU are not found in N2-fixing Paenibacillus strains, and the mechanisms involved in Fe-S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe-S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe-S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe-S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.

The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Synthesis of nitrogenase by Paenibacillus sabinae T27 in presence of high levels of ammonia during anaerobic fermentation.

Biological nitrogen fixation is usually inhibited by fixed nitrogen. Paenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph, possesses high nitrogenase activity and has 3 copies of nifH (nifH, nifH2, nifH3), a copy of nifDK, and multiple nifHDK-like genes. In this study, we found that P. sabinae T27 showed nitrogenase activities not only in low (0-3 mM) concentrations of NH4+ but also in high (30-300 mM) concentrations of NH4+, no matter whether this bacterium was grown in a flask or in a fermenter on scale cultivation. qRT-PCR and western blotting analyses supported that Fe protein and MoFe protein were synthesized under both low (0-3 mM) and high (30-300 mM) concentrations of NH4+. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that MoFe protein was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and nitrogen-excess conditions were active. This is the first time to report that diazotrophs show nitrogenase activity in presence of high (30-300 mM) concentrations of NH4+. Our study will provide a clue for studying the mechanisms of nitrogen fixation in presence of the high concentration of NH4+. KEY POINTS: • P. sabinae T27 can synthesize active nitrogenase in presence of high levels of ammonia. •Fe and MoFe proteins of nitrogenase purified in absence of ammonia are the same as those purified from the high concentration of ammonia. • Fe protein is encoded by nifH and nifH2, and MoFe protein is encoded by nifDK.

Diazotroph Paenibacillus triticisoli BJ-18 Drives the Variation in Bacterial, Diazotrophic and Fungal Communities in the Rhizosphere and Root/Shoot Endosphere of Maize.

Application of diazotrophs (N2-fixing microorganisms) can decrease the overuse of nitrogen (N) fertilizer. Until now, there are few studies on the effects of diazotroph application on microbial communities of major crops. In this study, the diazotrophic and endospore-forming Paenibacillus triticisoli BJ-18 was inoculated into maize soils containing different N levels. The effects of inoculation on the composition and abundance of the bacterial, diazotrophic and fungal communities in the rhizosphere and root/shoot endosphere of maize were evaluated by sequencing the 16S rRNA, nifH gene and ITS (Inter Transcribed Spacer) region. P. triticisoli BJ-18 survived and propagated in all the compartments of the maize rhizosphere, root and shoot. The abundances and diversities of the bacterial and diazotrophic communities in the rhizosphere were significantly higher than in both root and shoot endospheres. Each compartment of the rhizosphere, root and shoot had its specific bacterial and diazotrophic communities. Our results showed that inoculation reshaped the structures of the bacterial, diazotrophic and fungal communities in the maize rhizosphere and endosphere. Inoculation reduced the interactions of the bacteria and diazotrophs in the rhizosphere and endosphere, while it increased the fungal interactions. After inoculation, the abundances of Pseudomonas, Bacillus and Paenibacillus in all three compartments, Klebsiella in the rhizosphere and Paenibacillus in the root and shoot were significantly increased, while the abundances of Fusarium and Giberella were greatly reduced. Paenibacillus was significantly correlated with plant dry weight, nitrogenase, N2-fixing rate, P solubilization and other properties of the soil and plant.

Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions.

Exopolysaccharides (EPS) are of high significance in bacterial biofilm formation. However, the effects of EPS cluster(s) on biofilm formation in Paenibacillus species are little known. In this study, we have shown that Paenibacillus polymyxa WLY78, a N2-fixing bacterium, can form biofilm. EPS is the major component of the extracellular matrix. The genome of P. polymyxa WLY78 contains two putative gene clusters (designated pep-1 cluster and pep-2 cluster). The pep-1 cluster is composed of 12 putative genes (pepO-lytR) co-located in a 13 kb region. The pep-2 cluster contains 17 putative genes (pepA-pepN) organized as an operon in a 20 kb region. Mutation analysis reveals that the pep-2 cluster is involved in EPS biosynthesis and biofilm formation. Disruption of the pep-2 cluster also leads to the enhancement of motility and change of the colony morphology. In contrast, disruption of the pep-1 cluster does not affect EPS synthesis or biofilm formation. More importantly, the biofilm allowed P. polymyxa WLY78 to fix nitrogen in aerobic conditions, suggesting that biofilm may provide a microaerobic environment for nitrogenase synthesis and activity.

Differentially expressed circular RNAs in a murine asthma model.

Allergic asthma is one of the most common allergic diseases; however, the mechanisms underlying its development have yet to be fully elucidated. Although allergic diseases are inheritable, genetic variance alone cannot explain the notable increase in the prevalence of allergic diseases over a short period of time in recent decades. Recently, research focus has been shifting to epigenetic factors, such as non­coding RNAs. Circular RNAs (circRNAs) are involved in the pathogenesis of various diseases. The aim of the present study was to further elucidate the etiology of allergic asthma by analyzing aberrantly expressed circRNAs in a murine asthma model. A mouse model of house dust mite allergen­induced asthma was established, and the qualified libraries were sequenced using next­generation sequencing. The expression levels of circRNAs were validated by reverse transcription­quantitative PCR (RT­qPCR) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for biological pathway classification and enrichment analysis of the aberrantly expressed circRNAs. In addition, the interaction network of the differentially expressed circRNAs and microRNAs (miRNAs) was constructed using Cytoscape. By next­generation sequencing, a total of 150 circRNAs were revealed to be upregulated and 130 were downregulated in the murine asthma model group compared with in the control group. GO and KEGG analyses demonstrated that the differentially expressed circRNAs were mainly involved in processes such as 'autoimmune disease', 'cell adhesion molecules (CAMs)' and 'endocytosis', among others. The expression levels of six circRNAs, namely three upregulated (circ_0000909, circ_0000629 and circ_0000455) and three downregulated (circ_0001454, circ_0000723 and circ_0001389) circRNAs, were validated by RT­qPCR. In conclusion, the analyses suggested that circRNAs performed critical functions via endocytosis (such as macrophage endocytosis), cell adhesion molecules and lipid metabolism in allergic asthma. The interaction network revealed that certain miRNAs that may serve a role in asthma could be regulated by the differentially expressed circRNAs.

Siderophore and indolic acid production by Paenibacillus triticisoli BJ-18 and their plant growth-promoting and antimicrobe abilities.

Paenibacillus triticisoli BJ-18, a N2-fixing bacterium, is able to promote plant growth, but the secondary metabolites that may play a role in promoting plant growth have never been characterized. In this study, untargeted metabolomics profiling of P. triticisoli BJ-18 indicated the existence of 101 known compounds, including N2-acetyl ornithine, which is the precursor of siderophores, plant growth regulators such as trehalose 6-phosphate, betaine and trigonelline, and other bioactive molecules such as oxymatrine, diosmetin, luotonin A, (-)-caryophyllene oxide and tetrahydrocurcumin. In addition, six compounds were also isolated from P. triticisoli BJ-18 using a combination of silica gel chromatography, sephadex LH-20, octadecyl silane (ODS), and high-performance liquid chromatography (HPLC). The compound structures were further analyzed by Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), and Electronic Circular Dichroism (ECD). The six compounds included three classical siderophore fusarinines identified as deshydroxylferritriacetylfusigen, desferritriacetylfusigen, and triacetylfusigen, and three indolic acids identified as paenibacillic acid A, 3-indoleacetic acid (IAA), and 3-indolepropionic acid (IPA). Both deshydroxylferritriacetylfusigen and paenibacillic acid A have new structures. Fusarinines, which normally occur in fungi, were isolated from bacterium for the first time in this study. Both siderophores (compounds 1 and 2) showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis, but did not show obvious inhibitory activity against yeast Candida albicans, whereas triacetylfusigen (compound 3) showed no antibiosis activity against these test microorganisms. Paenibacillic acid A, IAA, and IPA were shown to promote the growth of plant shoots and roots, and paenibacillic acid A also showed antimicrobial activity against S. aureus. Our study demonstrates that siderophores and indolic acids may play an important role in plant growth promotion by P. triticisoli BJ-18.

Phosphate solubilizing bacteria stimulate wheat rhizosphere and endosphere biological nitrogen fixation by improving phosphorus content.

Phosphate (P) availability often limits biological nitrogen fixation (BNF) by diazotrophic bacteria. In soil, only 0.1% of the total P is available for plant uptake. P solubilizing bacteria can convert insoluble P to plant-available soluble P (ionic P and low molecular-weight organic P). However, limited information is available about the effects of synergistic application of diazotrophic bacteria and P solubilizing bacteria on the nitrogenase activity of rhizosphere and nifH expression of endosphere. In this study, we investigated the effects of co-inoculation with a diazotrophic bacterium (Paenibacillus beijingensis BJ-18) and a P-solubilizing bacterium (Paenibacillus sp. B1) on wheat growth, plant and soil total N, plant total P, soil available P, soil nitrogenase activity and the relative expression of nifH in plant tissues. Co-inoculation significantly increased plant biomass (length, fresh and dry weight) and plant N content (root: 27%, shoot: 30%) and P content (root: 63%, shoot: 30%). Co-inoculation also significantly increased soil total N (12%), available P (9%) and nitrogenase activity (69%) compared to P. beijingensis BJ-18 inoculation alone. Quantitative real-time PCR analysis showed co-inoculation doubled expression of nifH genes in shoots and roots. Soil nitrogenase activity and nifH expression within plant tissues correlated with P content of soil and plant tissues, which suggests solubilization of P by Paenibacillus sp. B1 increased N fixation in soils and the endosphere. In conclusion, P solubilizing bacteria generally improved soil available P and plant P uptake, and considerably stimulated BNF in the rhizosphere and endosphere of wheat seedlings.